Extraction of aureomycin



United States Patent nrxrsaciuouos AUREoMYolN Edward Everett Starbirgl, vNanuet, and Charles Pdacks,

Spring Valley, N. Y., assignors to American Cyan- -Cllmllll ,New Ynrk, N. Y., a corporation of ...eine i.

EN Drawmgf` Applicationseptember 12,1950, s erig11s0.1s4,s1s

4 claims. rc1. 'ero-ssc) materials not fsoluble in acidiied lower aliphatic-alcohols.'

Many ofthe impurities vwhich inthe past have been most diflcul-t to separate from aureomyciu are :not soluble in the -acidied lower aliphatic alcohols. aureomycin whether found associated with impurities as a ysalt with `an acid, the-Iree aureomycin, or the salt `with a metal. In the Yacidiiied 'lower aliphatic alcohol solution, the aureomycin probably exists as a salt with an acid, but it may be converted to any desired form during the process of its separation from the solvent, or later.

,This application is a ontinuation=inpart of application Serial Number 62,722 tiled November 30, 1,948, fen- Vtitled Isolation Vof Aureomycin, .and Vapplication Serial Numberv62,766 filed November, 1.948, entitled vIsolation of Antibiotic. Both applications are abandoned in favor of application, Serial Number 364,182,1iled June `2,5, flg953tffentitled .Chlortetracycline Puritication and Alkaline Ear-th Salts In Serial Nnnrberr62722frthere is disclosed at page k,6, lines 2 and 3 that aureomycin may be seperated from a .cake by the use .of solvents., listing among others, :the alcohols.

This applies tov 2,740,816 Patented A pr. 3, 19.55

ice

the strontium or magnesium salts, any 0r all ,of .which may be present In the presence of en excess of any of ythese metallic ions, a large proportion .of ,the aureomycin precipitates, perticulerlyif the PH of v the fermentation liquor is within the ,ronge of o to 10- 4The best recoverf ies usually occur if thev separation is conducted at a .pijl within the vrange of about 7 to 8.5. The precipitated aoreornyein may then be Separated troni the soluble Inaterials and such separation maybe aided by the use of a filter aid. The thus separated solids present the aureomyin-containing cake in an alkaline condition. The preparation of this cake is ,described in detail ,and claimed in our application Serial `Number 62,722.

An aureomycin-containing 'solid material may be obtained by precipitating the aureomycin from an aqueous solution v.containing"it by shitting the PH to `within .the Vrange of about v5 to 7, in which case it precipitates as neutral aureomycin which, of course, may contain certain impurities- .Other aureomycin-containing solids result from evaporation of a solution containing aureomycin, etc.

Any of these solids or .ca lgzes may then be treated in accordance with this invention .by suspending them in a lower aliphatic alcohol at a pH .of less than about A, whereby .the cnreornycin is -dissolved and the maior portion c f the impurities remain behind as insoluble A,materials The .ernennt of acid 4for the control of the PH varies to a large extent 4with the ,composition of the celte; and particularly .if linie .is Present, comparatively large quantities may be required.V 4The extraction may be .repeeterl- `Coonter-cnrrent .or multiple extraction. s may Weheve found that the lower aliphatic alcohols sive particularly .satisfactory results especially vwhen the cxtrection isfconslncted yet e PH o f less than ehol1t4- eohols with 2 to 5 .carbon etotns are preferred. .Oi the lower aliphatic alcohols nfbutnnol possesses y-v.l s l eti li.tv characteristics, solubility l.chen'icteristics and .c specific gravity which renders it peculiarly suitable for use as the extracting solvent. Additionally, aureomycin is comparatively :soluble in acidied rr-bntanoland many of the impnrities with which 'the eureorn-ycin has vpreviously been f ollllfl i9 Q SsSQsiie are comparatively insoluble in, this ac islied bunnol Qns n rocess the fused 4on .e vavide variety of materials luli-.ich nureomvein :is present. Our process rney yhe used in .coninnctien ithanyorall otseveralctherfpnriheationrrocedures each o f und iced ,materials with 4.rr/h ich the anreomycin .may Khe others. :one source Vof impure aureomvcin .is an alkaline .calce :which hes :been separated from the ferorientation arsch Many the fermentation procedures currently fhei seo result in fthe tforrnationrof In .final ligue;- 41u-.rvh1c;l 1 f the aureonrycin -is partially present in .fsfiltlin and .partiallypresent as a precipitate. If cal.- ciurn is fpresent, 4the precipitate :may be ythe calcium salt of aureomycin. 'Ether common -salts include weber-lum,

' ch separates .certain ,of the Y slower than in the preferred range.

he n sed tney various types of .extraction columns .and extraction equipment. The .extraction may be performed on the solids while wet- The .extraction may he Per.- orrned on edrv coke with rlrv butanol. Better recoverics are ,obtained if ,either the cake or the butanol is wet. In '.dry bntrnrolJ -eureonrvcin hydrochloride vis soluble vto the extent 0f vapprximavtely 500 gamrnas per cc. In wet butanol, it is soluble .to the extent of about 7000 gammas per l Surprisingly, it therefore appears that `if the wet calce is used, pot'only ,is the process simplified because it `is not necessary to dry the cake, but additionally a more efficient process is obtained. It is particularly conveulent .whenthe solids have been separated from. aqueous .solutions .to use thorn .wet and .thus save the cost of drying. .The extraction nray .be performed et any converricht .temperaturelenveen 10 .C- and 55 I C. fthe one n is. more .convenient- A smaller quantity of solvent ay'he used if the .extraction is performed 4 het and the solution .is less viscous .so .that separation vis easier- The extraction tney he ellciently performed et roorn tenineretnre though, thus-:saving l,the equipment necessary Ifor handling hot solvents Yerioos holds, .such .es hydrochloric, sulfuric, phosnhoricsacetic. .etctney he used for the pH control..4 Hvdrochloric and sulfuric ere the vrnost economical. We prefer ,the use ,of hydrochloric as Vthereby the aureomycin iney he nioreeasily Arecovered es the hydrochloride salt, but' sulfuric .seid is very economical. particularly if large amounts of lime must be neutralized, and the sulfate ,salt of aureomycin is easily :transformed to the hydrochloride during subsequent processing steps if desired.

We preferto 4rex-tract within the vpI-I ran-ge of about l to 2.51 More acid solutions require the use ofjlarger amounts of acid and more corrosion resistant'equipment without a commensurate increase lin yield. 'I f thevpljl is above .about 2.5, the extraction is not as complete and is l Any of the lower aliphatic alcohols may b'e used, but -butanol exhibits great solventpropertiesfor aureomycin in the acid ranges. It scheap, fis V leiss hazardousthaii some of the other 'ab cohols `from the standpoint of `fire, toxicity, and 'is easily recovered. It is desirable that an alcohol be used which may be removed from the extract at a temperature not in excess of about 55 C. and it is desirable that a solvent be used in which the impurities which occur with aureomycin are comparatively insoluble.

It is also preferred that one be used which is comparatively immiscible with water and one from which any residual water is preferentially removed during distillation. Normal butanol exhibits all of these characteristics.

The quantity of the solvents varies with the pH and the efiiciency of the solvent. At a pH within the preferred range, n-butanol gives excellent recoveries if two extractions are performed. On the recovery of aureomycin from fermentation cake, volumes equal to about 20% of the original mash volume for each extraction give an eiiicient recovery, The exact volume used and the number of extractions may be varied within wide limits.

The various extracts containing the aureomycin may then be combined and the aureomycin recovered therefrom. This is easily done if the alcohol is removed by distillation. To avoid damage to the aureomycin, it is preferred that the distillation be conducted below a maximum temperature of about 55 C. The alcohol distilled over may, of course, be re-used. The amount of water which will remain behind with thc alcohol varies, of course, with the alcohol being used. With n-butanol, the water distills over as an azeotrope leaving as the residual solvent dry butanol. The acidity of the residue depends in part upon the acid being used. If hydrochloric acid is used, some of the acid may distill over and additional acid may be required to assist in the crystallization of the aureomycin.

Generally, the solvent is distilled off until the aureomycin is at least suiiiciently concentrated to precipitate when the concentrate is cooled. We have found improved recoveries occur if the solvent is distilled over until the aureomycin starts to precipitate out. while hot and may be continued until a fairly thick slurry is formed. As

a matter of operating convenience, we find it convenient to concentrate the solvent to about V20 of its original volume. The slurry may then be chilled and the aureomycin separated out. For best recoveries it appears desirable that the slurry be rather acid at this point and it may well be adjusted to a pH of about 0.8 by the addition of hydrochloric acid.

After chilling, the crystals may be separated, then washed and dried. The washes for the crystals may be at least one from the group consisting of n-butanol, ethyl Cellosolve, ethyl alcohol, and water. Best results are ob- V tained when at least two of these solvents in succession are used to wash the crystals. It is usually desirable that the subsequent wash be performed before the crystals dry.

if an acid other than hydrochloric is used for the acidification in the original extraction, the acid radical may be changed by shaking the extract with a suitable salt. If sulfuric acid is used in the original extraction and the solvent used is butanol, a saturated sodium chloride wash may be shaken with the solvent which will result in a change of the aureomycin in solution from the sulfate to the hydrochloride salt. Other changes may be made if desired. The aureomycin is usually desired as the hydrochloride.

Example 1 A cake containing aureomycin from the filtration with alkaline fermentation liquor was treated with 20% by volume of the original mash liquor of n-butanol at room temperature at a pH of 1.4 being made acid with 9 N sulfuric acid. The butanol cake and acid were slurried together for ten minutes after the desired pH was reached and then the cake was filtered. The resultant cake was again slurried with a 20% by volume of butanol for ten minutes at a pH of 1.4 and the butanol and cake separated. The first extract contained 64.8% of the aureomycin originally present and the second extraction, 18%. The butanolic extracts were pooled and shaken with 30% sodium chloride solution. The upper solvent phase was separated and the mixture concentrated under vacuum until the aureomycin began to pricipitate, after which it was chilled, adjusted to a pH of 0.8 with hydrochloric acid and the aureomycin separated by filtration. The rcsultant product was the hydrochloric acid salt of aureomycin.

Example 2 9.5 liters oi an aureomycin fermentation mash assaying 1185 gammas per cc. had addedthereto 1% on a weight by volume basis of diatomaceous earth. The mash was filtered, giving approximately 1 kilogram of a wet cake. 'lhe cake was extracted with 1.9 liters of anhydrous butanol, the butanol separated, and the residue again extracted wlih 1.9 liters ot' wet butanol. This last extraction was repeated. The first extraction was performed at a pH of 1.30 which required the addition of approximately 170 cc. of 6 normal hydrochloric acid. The second and third extractions were performed after the addition of 13 cc. of 6 normal hydrochloric acid with the pHs of 1.46 and 1.35 respectively. The aureomycin analyses on the three batches were, for the first extraction 1760 cc. of extract at 5690 gammas per cc. The second, 1920 cc. at 910 gammas per cc. The third, 1929 cc. at 220 gammas per cc. (Due to uncertainties in the analyses there is not obtained an exact balance.) The butanol extracts were pooled and the butanol removed under vacuum at a temperature between and 55 C. to approximately 5% of the original butanol volume. To the concentrate was added 1.10 cc. of Cellosolve and 13 cc. of 6 normal hydrochloric acid. The concentrate was allowed to age at room temperature for 18 hours and for 7 hours at 4 C. The material was filtered, washed with 15 cc. of Cellosolve, 12 cc. of water, 12 cc. of anhydrous alcohol, and then dried.

Example 3 To 12 liters of mash at a pH of 7.2 was added 24 cc. of 10 normal sodium hydroxide and 180 grams of siliceous earth. The resulting slurry was filtered, yielding 1350 grams of a wet mycelial cake. A 200 gram portion of this cake was extracted twice with 360 cc. of butanol, the first extract, dry butanol, and the second extract, wet. The first extraction was performed at a pH of 0.38 by the addition of 65 cc. of concentrated hydrochloric acid, and the second at a pH of 0.32 by the addition of 30 cc. of concentrated hydrochloric acid. The extracts were pooled, the aqueous layer discarded and the extract concentrated to 40 cc. under vacuum at a temperature between 45 and C. To the concentrate was added 16 cc. of Cell0- solve, the mixture permitted to stand for 18 hours at room temperature, then 4 hours at 4" C. The crystals were separated and washed with 4 cc. of Cellosolve, 3 cc. of water and 3 cc. of anhydrous alcohol, and dried.

Example 4 1.9 liters of a mash assaying 940 gammas per cc. were adjusted to a pH of 8.6 with l0 normal caustic soda. To this was added 28 grams of diatomaceous earth, and the mixture filtered. The cake was made acid with 52 cc. of 6 normal hydrochloric acid and was extracted once with 450 cc. of isopropyl alcohol. The solids were again extracted with 450 cc. of isopropyl alcohol after the addition of 7 cc. of 6 normal hydrochloric acid. Both extractions were performed at r55 C. The resulting extracts were pooled and concentrated to 10% of their original volume in vacuo at 35 C. To the concentrate was added 1/5 of its volume of ethyl Cellosolve and its own volume of anhydrous alcohol. The pH was adjusted to 0.69 with 6 cc. of 6 normal hydrochloric acid. 'Ihe concentrate was permitted to stand-at room temperature for hours and at 4 C. for 4 hours. The material was filtered, washed once with 4 cc. of Cellosolve, then 4 cc. ofwater and finally with 4 cc. of anhydrous ethyl a1- cohol. The crystals were dried in vacuo. There was obtained a yield of 1.06 grams of aureomycin hydrochloride assaying 720 gammes per milligram which is a recovery of 38.4%

Example 5 40 liters of mash assaying 1120 gammas per cc. were adjusted to a pH of 8.45 with 53 cc. of l0 normal sodium hydroxide. 800 grams of Hy-Flo Supercel were added and the mixture iltered, yielding 5147 grams of aureomycin cake.

250 grams of the above cake, wet, representing 1945 cc. of the mash, were extracted with 350 cc. of secondary butanol after the addition of 40 cc. of 6 normal hydrochloric acid, the extraction being at a pH of 1.21, there being obtained 366 cc. of an extract assaying 4310 gammas per cc. A second extraction was performed using 485 cc. of secondary butanol and 3 cc. of 6 normal hydrochloric acid at a pH of 1.43, yielding 398 cc. of an extract assaying 690 gammas per cc. The aqueous layer was separated and the butanol extracts combined. The material was concentrated to 5% of its original volume in vacuo at a temperature of 35 to 40 C. To the concentrate was added 0.5 cc. of 6 normal hydrochloric acid, giving a pH of 0.50. The concentrate was permitted to age 18 hours at room temperature and 24 hours at 4 C. The crystals were filtered, washed with 4 cc. of Cellosolve, 4 cc. of water, and 4 cc. of anhydrous alcohol, and dried in vacuo.

Example 6 l2 liters of aureomycin fermentation mash had added thereto sufficient calcium hydroxide to raise the pH to 7.5, then the mixture was ltered. The cake. was suspended in 2.4 liters of normal butanol at a pH of approximately 1.4 using approximately 160 cc. of 6 normal hydrochloric acid for pH control. The butanol cake slurry was stirred for 10 minutes after the desired pH was reached and then filtered. The resultant cake was again slurried with 2.4 liters of normal butanol at the same pH. Approximately cc. of hydrochloric acid was required to maintain the required pH. An aqueous layer resulting from the rst extraction was separated from the butanol and discarded as it contained but a small amount of aureomycin. The butanolic extracts were pooled and the butanol partially removed therefrom in a vacuum still. Approximately 450 cc. were retained. The temperature in the still was kept under 55 C. during the entire concentration. The concentrated mixture in which the aureomycin was beginning to precipitate was adjusted to a pH of approximately 0.8 with 1:1 hydrochloric acid, approximately 40 cc. being required. The concentrated butanol was allowed to age for 12 hours at room temperature and then chilled for 6 hours. The crystals were removed by filtration, slurried, and removed from each of Cellosolve, water, and anhydrous ethyl alcohol. The crystals were dried at 40 C.

Example 7 7.15 liters of aureomycin mash at a pH of 6.19 were treated with 8.4 cc. of 10 normal sodium hydroxide, and 215 grams of Hy-Flo Supercel added thereto. The mash was filtered, yielding 786 grams of a wet alkaline cake.

This cake was extracted twice with 1570 cc. each of dry normal butanol, at a pH of 1.26 each. 'Ihe iirst required 113 cc. of 6 normal hydrochloric acid and the second 5 cc. of 6 normal hydrochloric acid. The butanol layers were separated from the aqueous layers, pooled and concentrated to 250 cc. in vacuo at a maximum temperature of 60 C. 250 cc. of anhydrous ethanol and 50 cc. of Cellosolve were added, and the crystals were aged two hours at room temperature and overnight at 4 C., ltered and washed rst with 5 cc. of butanol, 3 times with 5 cc. each of Cellosolve and then 5 cc. of water and inally 10 cc. of ethyl alcohol. The

crystals were dried, yielding 6.12 grams of aureomycin hydrochloride with a purity of 580 gammas per milligram, a yield of 55.2% based on the mash activity.

Variations in the above procedures, and minor moditcations, will suggest themselves to those skilled in the art.

As our invention we claim:

1. A process for the purification of chlortetracycline which comprises the steps of extracting at a pH less than about 4 the water-wet solids which are precipitated from an aqueous fermentation liquor containing chlortetracycline at a pH within the range 6 to l0 with a saturated, nnsubstituted lower aliphatic alcohol having from 2 to 5 carbon atoms to dissolve the chlortetracycline contained therein, separating the alcoholic solution from insoluble matter, removing water from said alcoholic solution and concentrating said solution by evaporation at a temperature less than about 55 C. until the chlortetracycline contained therein precipitates.

2. A process for the purification of chlortetracyclinc which comprises the steps of extracting at a pH less than about 4 the water-wet solids which are precipitated from an aqueous fermentation liquor containing chlortetracycline at a pH within the range 6 to 10 with n-butanol todissolve the chlortetracycline contained therein, separating the alcoholic solution from insoluble matter, removing water from said alcoholic solution and concentrating said solution by evaporation at a temperature less than about 55 C. until the chlortetracycline contained therein precipitates.

3. A process for the purification of chlortetracycline which comprises the steps of extracting at a pH less than about 4 the water-Wet solids which are precipitated from an aqueous fermentation liquor containing chlortetracycline at a pH within the range 6 to l0 with a saturated, unsubstituted lower aliphatic alcohol having from 2 to 5 carbon atoms to dissolve the chlortetracycline contained therein, separating the alcoholic solution from insoluble matter, removing water from said alcoholic solution and concentrating said solution by evaporation at a temperature less than about 55 C. adding hydrochloric acid to said concentrated solution and allowing the mixture to stand at lower temperature until the chlortetracycline is precipitated as its hydrochloric acid salt.

4. A process for the purification of chlortetracycline which comprises the steps of extracting at a pH less than about 4 the Water-wet solids which are precipitated from an aqueous fermentation liquor containing chlortetracycline at a pH within the range 6 to 10 with a saturated, unsubstituted lower aliphatic alcohol having from 2 to 5 carbon atoms to dissolve the chlortetracycline contained therein, separating the alcohol solution from insoluble matter, removing water from said alcoholic solution and concentrating said solution by evaporation at a temperature less than about 55 C. adding ethoxyethanol to said concentrated solution, allowing the mixture to stand and thereafter recovering the product that crystallizes therefrom.

References Cited in the le of this patent UNITED STATES PATENTS 2,461,922 Rake Feb. l5, 1949 2,482,055 Duggar Sept. 13, 1949 2,516,080 Sobin July 18, 1950 2,586,766 Pidacks et al. Feb. 19, 1952 2,655,535 Pidacks et al Oct. 13, 1953 OTHER REFERENCES .Application No. 83,780, Abstracted in 650 O. G. 895, September 18, 1951.

Broschard in Science 109: pp. 199-200, February 25, 1949.

Brook in I. Biol. Chem., vol. 165, October 1946, pp. 463-68. 

1. A PROCESS FOR THE PURIFICATION OF CHLORTETRACYCLINE WHICH COMPRISES THE STEPS OF EXTRACTING AT A PH LESS THAN ABOUT 4 THE WATER-WET SOLIDS WHICH ARE PRECIPITATED FROM AN AQUEOUS FERMENTATION LIQUOR CONTAINING CHLORTETRACYCLINE AT A PH WITHIN THE RANGE 6 TO 10 WITH A SATURATED, UNSUBSTITUTED LOWER ALIPHATIC ALCOHOL HAVING FROM 2 TO 5 CARBON ATOMS TO DISSOLVE THE CHLORTETRACYCLINE CONTAINED THEREIN, SEPARATING THE ALCOHOLIC SOLUTION FROM INSOLUBLE MATTER, REMOVING WATER FROM SAID ALCOHOLIC SOLUTION AND CONCENTRATING SAID SOLUTION BY EVAPORATON AT A TEMPERATURE LESS THAN ABOUT 55* C. UNTIL THE CHLORTETRACYCLINE CONTAINED THEREIN PRECIPITATES. 